Methods for codon optimization and uses thereof

Abstract

Provided herein are methods and systems for codon rewriting and replacement. In some aspects, provided herein, is a method comprising: analyzing at least a portion of a genome of an organism to identify a first plurality of codons based on at least in part on a first local context of a codon-of-interest in the genome of the organism to be rewritten; and rewriting the first plurality of codons in the genome of the organism to a second codon. Also provided herein are methods and systems for producing a synthetic genome.

1 . A method comprising:
 a) analyzing at least a portion of a genome of an organism to identify a first plurality of codons based on at least in part on a first local context of a codon-of-interest in the genome of the organism to be rewritten;   b) rewriting the first plurality of codons in the genome of the organism to a second codon, wherein the first plurality of codons and the second codon encode a first amino acid, and wherein the rewriting of the first plurality of codons modulates an occurrence of the first plurality of codons;   c) synthesizing a nucleic acid construct comprising the portion of the genome of the organism, wherein the first plurality of codons is rewritten to the second codon; and   d) introducing the nucleic acid construct into a cell of the organism to replace the portion of the genome of the organism.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , wherein the modulating of the occurrence of the first plurality of codons comprises eliminating the occurrence of the first plurality of codons. 
     
     
         4 . The method of  claim 1 , wherein the analyzing comprises identifying one or more synonymous codons with a least number of occurrences in the genome of the organism, wherein the first plurality of codons comprises the one or more synonymous codons with the least number of occurrences. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 4 , wherein the analyzing further comprises determining a number of occurrences of the first local context of the codon-of-interest, wherein the first local context of the codon-of-interest comprises
     C   (n−1)   −C   n   −C   (n+1) , wherein   C (n−1)  denotes a codon downstream of the codon-of-interest;   C n  denotes the codon-of-interest; and   C (n+1)  denotes a codon upstream of the codon-of-interest.   
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 6 , the preceding, wherein the analyzing further comprises determining a relative synonymous codon usage (RSCU) of the codon-of-interest. 
     
     
         9 . The method of  claim 8 , wherein the analyzing further comprises identifying the first plurality of codons based at least in part on a second local context of the codon-of-interest in the genome of the organism, wherein the second local context of the codon-of-interest comprises
     C   (n−1)   −AA   n   −C   (n+1) , wherein   C (n−1)  denotes a codon downstream of the codon-of-interest;   AA n  denotes an amino acid encoded by the codon-of-interest; and   C (n+1)  denotes a codon upstream of the codon-of-interest.   
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 9 , wherein the analyzing further comprises determining a number of occurrences of the second local context of the codon-of-interest. 
     
     
         12 . The method of  claim 11 , wherein the analyzing further comprises determining an expected number of occurrences of the first local context of the codon-of-interest, wherein the expected number of occurrences of the first local context of the codon-of-interest is determined as a product of: a number of occurrences of the second local context of the codon-of-interest, and the determined RCSU of the codon-of-interest. 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the analyzing comprises processing the at least the portion of the genome of the organism using a machine learning-based computer system, wherein the machine learning-based computer system comprises one or more storage units comprising, respectively, one or more storage devices included within respective storage arrays controlled by a respective one or more storage controllers; and one or more computer processing units, wherein the one or more computer processing units communicate with the one or more storage units over a communication interface. 
     
     
         15 . (canceled) 
     
     
         16 . The method of  claim 1 , wherein the analyzing further comprises identifying one or more statistically significant evolutionary signals, wherein the one or more statistically significant evolutionary signals comprise a negative evolutionary selection signal, a positive evolutionary selection signal, or a combination thereof;
 wherein the negative selection signal comprises a frameshift, a ribosome stall, or a secondary RNA structure interfering with transcription or translation; and   wherein the positive selection signal comprises a regulatory element within an open reading frame (ORF).   
     
     
         17 .- 19 . (canceled) 
     
     
         20 . The method of  claim 1 , wherein the method further comprises reassigning the first plurality of codons to a second amino acid. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 1 , wherein the first amino acid comprises arginine, leucine, or serine. 
     
     
         23 . (canceled) 
     
     
         24 . The method of  claim 1 , wherein the first plurality of codons comprises CGA, CGG, or a combination thereof. 
     
     
         25 . (canceled) 
     
     
         26 . The method of  claim 1 , wherein the first plurality of codons comprises CTA, CTG, or a combination thereof. 
     
     
         27 . (canceled) 
     
     
         28 . The method of  claim 1 , wherein the first plurality of codons comprises AGT, AGC, TCG, TCA, or a combination thereof. 
     
     
         29 . The method of  claim 1 , wherein the rewriting further comprises removing a plurality of tRNA molecules with anticodons that recognize the first plurality of codons, wherein the removing comprises deleting one or more genes that encode the plurality of tRNA molecules that recognize the first plurality of codons. 
     
     
         30 . (canceled) 
     
     
         31 . The method of  claim 20 , further comprising providing the cell (i) additional tRNA molecules that recognize the first plurality of codons and aminoacyl-tRNA synthetases (aaRSs) for charging the additional tRNA molecules with the second amino acid: (ii) a tRNA pre-charged with the second amino acid: or (iii) both (i) and (ii). 
     
     
         32 . (canceled) 
     
     
         33 . The method of  claim 20 , wherein the second amino acid comprises a non-canonical amino acid, wherein the non-canonical amino acid comprises an azide-containing ncAA, an alkene-containing ncAA, an alkyne-containing ncAA, p-azidophenylalanine, 2-aminoisobutyric acid (Aib), N6-[(propargyloxy)carbonyl]-L-lysine, O—4-allyl-L-tyrosine, or a combination thereof. 
     
     
         34 . (canceled) 
     
     
         35 . The method of  claim 1 , wherein the rewriting of the first plurality of codons comprises modulating one or more codons in the first plurality of codons, wherein the one or more codons are within 4 codons of each other. 
     
     
         36 . The method of  claim 1 , wherein the rewriting of the first plurality of codons comprises modulating a codon fragment of one or more codons in the first plurality of codons, wherein the codon fragment comprises a trimer, a hexamer, a 9 mer, or a combination thereof. 
     
     
         37 . (canceled) 
     
     
         38 . A method of producing a polypeptide comprising a non-canonical amino acid (ncAA) or a population of polypeptide molecules comprising the ncAA in an organism, the method comprising:
 rewriting a first codon encoding a first amino acid to a second codon encoding the first amino acid in a genome of the organism, wherein the rewriting comprises identifying the first codon based at least in part on a first local context of a codon-of-interest in the genome of the organism;   reassigning the first codon to encode the ncAA in the genome of the organism; and   introducing into the organism an aminoacyl-tRNA synthetase (aaRS)/tRNA pair engineered to recognize the first codon and incorporate the ncAA into an amino acid sequence of the polypeptide or the population of the polypeptide molecules.   
     
     
         39 .- 67 . (canceled) 
     
     
         68 . A cell or a population of cells comprising a genome, wherein a first plurality of codons in the genome of the organism is rewritten to a second codon, wherein the first plurality of codons and the second codon encode a first amino acid, and wherein an occurrence of the first plurality of codons is modulated responsive to being rewritten to the second codon, wherein the first plurality of codons is reassigned to a second amino acid. 
     
     
         69 .- 103 . (canceled)